This invention relates generally to an antigenic preparation and specifically to Leptospira membrane proteins which are used to induce a protective immune response in animals. Such proteins can be used immunologically as vaccines for leptospirosis caused by this organism. Alternatively, diagnosis of leptospirosis can be performed by detecting the presence of the proteins, antibodies to the proteins, or polynucleotides which encode the proteins.
Leptospirosis is an important, global human and veterinary health problem. It is a widespread zoonotic disease caused by pathogenic strains of Leptospira which are capable of infecting most mammalian species. Infection occurs either through direct contact with an infected animal or indirect contact with contaminated soil or water. In livestock, the disease causes economic losses due to abortion, stillbirth, infertility, decreased milk production, and death.
Efforts to control leptospirosis have been hampered because virulent leptospiras have the capacity for both long-term survival in the environment as well as persistent infection and shedding by wildlife and livestock. Currently available leptospiral vaccines produce short-term immunity and do not provide cross-protection against many of the 170 serovars of pathogenic Leptospira {Thiermann, et al., J. Am. Vet. Med. Assoc., 184:722 (1984)}. These vaccines consist of inactivated whole organisms or outer envelope preparations which produce seroreactivity as determined by microscopic agglutination of intact organisms. The nature of the protective immunogens in these vaccine preparations has not been conclusively elucidated, although several lines of evidence suggest that lipopolysaccharide-like substance (LLS) may confer a degree of protection. Commercially available vaccines, which consist of heat or formalin-killed leptospiras, produce incomplete or only short-term immunity, requiring their administration annually or semi-annually. In the case of L. interrogans serovar hardjo, the common bovine pathogen in North America, vaccines prepared in this way are ineffective {Bolin, C. A., et al., Am. J Vet. Res., 50:161-165 (1989) and Bolin, C. A., et al., Am. J. Vet. Res., 50:2004-2008 (1989)}. Thus there is an important need for development of an improved leptospiral vaccine.
The pathogenesis of leptospirosis is very similar to that of other spirochetal diseases, including syphilis (caused by Treponema pallidum) and Lyme borreliosis (caused by Borrelia burgdorferi). Both syphilis and Lyme borreliosis are characterized by widespread dissemination early in the course of disease, including invasion of the central nervous system. Leptospira share this ability with other pathogenic spirochetes such that meningitis is a common manifestation of leptospirosis. Another feature of spirochetal infections is the ability to persist chronically in the host, as manifested in cases of tertiary syphilis and chronic Lyme arthritis.
Lipid-modified, integral membrane proteins have been identified in a broad range of bacterial species {Hayashi, S., et al., J. Bioenerg. Biomembr., 22:451-471 (1990)}. In gram-negative bacteria, these lipoproteins are processed by signal peptidase II {Pugsley, A. P., Microbiol. Rev., 57:50-108 (1993)} after covalent linkage of three fatty acid residues to an N-terminal cysteine {Hantke, et al., Eur. J. Biochem., 34:384-296 (1973)}. The fatty acid residues anchor the lipoproteins to either the cytoplasmic membrane or the outer membrane. Although the polypeptide portion of lipoproteins is generally hydrophilic, lipid modification renders them amphiphilic and causes them to partition into the hydrophobic phase during Triton X-114 phase partitioning {Chamberlain, N. R., et al., Infect. Immun., 57:2872-2877 (1989)}.
Lipoproteins have been identified in a number of spirochetes including, Treponema pallidum {Chamberlain, N. R., et al., Infect. Immun., 57:2872-2877 (1989) and Chamberlain, N. R., et al., Infect. Immun., 57:2878-2885 (1989)}, Treponema denticola {Miyamoto, M., et al., Infect. Immun., 59:1941-1947 (1991)}, Serpulina hyodysenteriae {Thomas, W., et al., Infect. Immun., 61:1136-1140 (1993)}, Borrelia burgdorferi {Brandt, et al., Infect. Immun., 58:983-991 (1990)}, and the relapsing-fever Borreliae {Burman, N., et al., Mol. Microbiol., 4:1715-1726 (1990)}. The lipoproteins appear to play an important role in the pathogenesis of spirochetal diseases. For example, many of the T. pallidum lipoproteins are immunodominant antigens, eliciting a strong humoral and cellular immune response {Akins, D. R., et al., Infect. Immun., 61:1202-1210 (1993)}. In addition, Outer Surface Protein A (OspA), of Borrelia burgdorferi is immunoprotective in animal models of Lyme disease {Fikrig, E., et al., Science, 250:553-556 (1990)}.
Triton X-114 solubilized material from both virulent and attenuated L. kirschneri (formerly L. alstoni and L. interrogans) strains partitioned into the hydrophobic detergent phase, and contained lipopolysaccharide like substance (LLS) from the organisms"" outer membrane components {Haake, D. A., et al., Infection and Immunity, 59:1131-40 (1991)}. In the study, the virulent strain of L. kirschneri contained greater amounts of an LLS component with an apparent molecular mass of 30 kilodalton (kDa). A later Haake, D. A., et al. publication discloses the cloning and sequencing of a gene encoding the OmpL1 (with a predicted molecular weight of 31,113 Da) protein of pathogenic Leptospira spp {Haake, D. A., et al., J. Bacteriol., 175:4225-4234 (1993)}. This might be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.
Unsuccessful research on the identification of Leptospira and T. pallidum OMPs has shown the importance of taking into account spirochetal outer membrane fragility and the lack of outer membrane selectivity of ionic detergents such as sodium dodecyl sulfate (SDS) {Cunningham, et al., J. Bacteriol., 170:5789 (1988); Penn, et al., J. Gen. Microbiol., 131:2349 (1985); Stamm, et al., Infect. Immun., 55:2255 (1987)}. Outer membrane proteins are of great importance because they play a key role in bacterial pathogenesis. The identification of outer membrane proteins involved in Leptospira pathogenesis is significant to understanding not only leptospiral outer membrane proteins and their involvement in pathogenesis, but also to understanding other spirochetal outer membrane proteins and their role in pathogenesis.
The present invention presents two novel leptospiral membrane proteins: LipL1 and LipL2. In particular, these proteins are lipoproteins which are associated with pathogenic strains of Leptospira. LipL1 is about 35 kDa, and LipL2 is about 41 kDa. Also disclosed are the method for purifying these proteins from Leptospira, their nucleotide and amino acid sequences, the cloning of the genes encoding the proteins and their recombinant proteins, methods for producing antibodies to these proteins, and the resulting antibodies. These proteins, their immunogenic fragments, and antibodies capable of binding to them, are useful for inducing an immune response to pathogenic Leptospira as well as providing a diagnostic target for leptospirosis.